A Putative Extracellular Salt Bridge at the Subunit Interface Contributes to the Ion Channel Function of the ATP-gated P2X2 Receptor

Jiang, Ruotian; Martz, Adeline; Gonin, Sophie; Taly, Antoine; Carvalho, Lia Prado de; Grütter, Thomas

doi:10.1074/jbc.m110.101980

Cited by 46 publications

(72 citation statements)

References 40 publications

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“…The holding potential was −60 mV. Dose-response relationship experiments and drug applications were carried out as described previously (5). The apparent reaction rate constant k app was determined by fitting data with a single exponential equation.…”

Section: Methodsmentioning

confidence: 99%

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Agonist trapped in ATP-binding sites of the P2X2 receptor

Jiang

Lemoine

Martz

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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ATP-gated P2X receptors are trimeric ion channels, as recently confirmed by X-ray crystallography. However, the structure was solved without ATP and even though extracellular intersubunit cavities surrounded by conserved amino acid residues previously shown to be important for ATP function were proposed to house ATP, the localization of the ATP sites remains elusive. Here we localize the ATP-binding sites by creating, through a proximity-dependent "tethering" reaction, covalent bonds between a synthesized ATPderived thiol-reactive P2X2 agonist (NCS-ATP) and single cysteine mutants engineered in the putative binding cavities of the P2X2 receptor. By combining whole-cell and single-channel recordings, we report that NCS-ATP covalently and specifically labels two previously unidentified positions N140 and L186 from two adjacent subunits separated by about 18 Å in a P2X2 closed state homology model, suggesting the existence of at least two binding modes. Tethering reaction at both positions primes subsequent agonist binding, yet with distinct functional consequences. Labeling of one position impedes subsequent ATP function, which results in inefficient gating, whereas tethering of the other position, although failing to produce gating by itself, enhances subsequent ATP function. Our results thus define a large and dynamic intersubunit ATPbinding pocket and suggest that receptors trapped in covalently agonist-bound states differ in their ability to gate the ion channel.affinity labeling | chemical modification | purinergic receptor P 2X receptors are oligomeric ATP-gated ion channels selective to cations (1) and are involved in physiological processes as diverse as synaptic transmission, the response to inflammation, and pain perception (2). Upon ATP binding, structural rearrangements of the subunit interface (3-5) lead to the opening of the ion channel (6-8), but the entire molecular sequence of events that couple ATP binding to channel opening remains unknown. The recent X-ray structure of the P2X4 receptor in a closed resting state represents in this regard a decisive step (9). It confirms the trimeric stoichiometry of the ion channel, in agreement with the fact that there are three activatable ATP-binding sites (10), and provides a structural context to interpret functional data (11).Early studies based on mutagenesis data have identified highly conserved extracellular residues important for ATP function and have proposed that ATP binding occurs through the extracellular domain (12-19), presumably at the subunit interface (15,17). When mapped on the crystal structure, most of these residues are observed to line a large and deep intersubunit cavity shaped like an open "jaw" and located approximately 45 Å away from the ion channel domain (9). This observation thus suggests that these residues participate in ATP binding; however, the crystal structure was solved in the absence of ATP, and therefore no direct evidence support this hypothesis to date.We used the proximity-dependent "tethering" approach (20) to localiz...

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Section: Methodsmentioning

confidence: 99%

“…Upon ATP binding, structural rearrangements of the subunit interface (3)(4)(5) lead to the opening of the ion channel (6)(7)(8), but the entire molecular sequence of events that couple ATP binding to channel opening remains unknown. The recent X-ray structure of the P2X4 receptor in a closed resting state represents in this regard a decisive step (9).…”

mentioning

confidence: 99%

Agonist trapped in ATP-binding sites of the P2X2 receptor

Jiang

Lemoine

Martz

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…These findings confirm the essential role of the lower body domain in conformational transitions during channel gating. D197 (rat P2X7, rP2X7 numbering), located in the lower body domain, is pivotal for acidic pH-induced channel inhibition [80] , suggesting an additional function of the lower body domain that is independent to the movement of the LF and DF domains, and this was confirmed by the importance of the salt bridge E63-R274 (rP2X2 numbering) in this domain for the channel gating [81] . Actually, both the closure of binding site jaw and the movement of the lower body domain are required for the concomitant pore opening of P2X receptors [33] .…”

Section: Lower Body Domainmentioning

confidence: 62%

Insights into the channel gating of P2X receptors from structures, dynamics and small molecules

Wang

2016

Acta Pharmacol Sin

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P2X receptors, as ATP-gated non-selective trimeric ion channels, are permeable to Na + , K + and Ca 2+ . Comparing with other ligand-gated ion channel families, P2X receptors are distinct in their unique gating properties and pathophysiological roles, and have attracted attention as promising drug targets for a variety of diseases, such as neuropathic pain, multiple sclerosis, rheumatoid arthritis and thrombus. Several small molecule inhibitors for distinct P2X subtypes have entered into clinical trials. However, many questions regarding the gating mechanism of P2X remain unsolved. The structural determinations of P2X receptors at the resting and ATPbound open states revealed that P2X receptor gating is a cooperative allosteric process involving multiple domains, which marks the beginning of the post-structure era of P2X research at atomic level. Here, we review the current knowledge on the structure-function relationship of P2X receptors, depict the whole picture of allosteric changes during the channel gating, and summarize the active sites that may contribute to new strategies for developing novel allosteric drugs targeting P2X receptors.

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“…Previous studies demonstrated that the ion channel function could be restored by charge swapping or disulfide trapping of the salt bridge in some receptors (31,32,35). Here, disruption of the salt bridge by charge reversal at one amino acid of the salt bridge attenuated ATP-induced maximum currents (I max ϭ 5.46 Ϯ 2.6 and 2.69 Ϯ 0.7 pA/pF, for R309D and D85R, respectively; n ϭ 6 -10; Fig.…”

Section: Salt Bridge Between Arg-309 and Asp-85 Is A Stringent Structmentioning

confidence: 92%

A Highly Conserved Salt Bridge Stabilizes the Kinked Conformation of β2,3-Sheet Essential for Channel Function of P2X4 Receptors

Zhao

Sun

et al. 2016

Journal of Biological Chemistry

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Significant progress has been made in understanding the roles of crucial residues/motifs in the channel function of P2X receptors during the pre-structure era. The recent structural determination of P2X receptors allows us to reevaluate the role of those residues/motifs. Residues Arg-309 and Asp-85 (rat P2X4 numbering) are highly conserved throughout the P2X family and were involved in loss-of-function polymorphism in human P2X receptors. Previous studies proposed that they participated in direct ATP binding. However, the crystal structure of P2X demonstrated that those two residues form an intersubunit salt bridge located far away from the ATP-binding site. Therefore, it is necessary to reevaluate the role of this salt bridge in P2X receptors. Here, we suggest the crucial role of this structural element both in protein stability and in channel gating rather than direct ATP interaction and channel assembly. Combining mutagenesis, charge swap, and disulfide cross-linking, we revealed the stringent requirement of this salt bridge in normal P2X4 channel function. This salt bridge may contribute to stabilizing the bending conformation of the ␤2,3-sheet that is structurally coupled with this salt bridge and the ␣2-helix.

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A Putative Extracellular Salt Bridge at the Subunit Interface Contributes to the Ion Channel Function of the ATP-gated P2X2 Receptor

Cited by 46 publications

References 40 publications

Agonist trapped in ATP-binding sites of the P2X2 receptor

Agonist trapped in ATP-binding sites of the P2X2 receptor

Insights into the channel gating of P2X receptors from structures, dynamics and small molecules

A Highly Conserved Salt Bridge Stabilizes the Kinked Conformation of β2,3-Sheet Essential for Channel Function of P2X4 Receptors

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